1. Field of the Invention
The present invention relates to a compound labelled by an enzyme, its preparation process and its use as a tracer or marker in enzymoimmunological determinations, e.g. in the form of a labelled antigen, labelled hapten or labelled antibody.
2. Discussion of the Background
It is known that in a higher vertebrate, an antigen can induce the production of specific antibodies able to form a reversible complex having a high bonding energy with the antigen. Haptens are low molecular weight substances, which do not bring about the formation of antibodies when injected into an animal, but which instead react with the antibodies. These antibodies directed against haptens can be produced on injecting into an animal or man a conjugate compound, e.g. of hapten and a protein.
The antigen--antibody or hapten--antibody reactions can be used for measuring the concentrations of haptens, antigens or antibodies in a given medium using immunoanalytical methods, such as enzymoimmunological methods. In the latter case, the reaction involves an antibody, an antigen or a hapten and a molecule labelled by an enzyme, which can either be an antigen, or the antibody, or the hapten.
As a function of the nature of the molecule labelled by the enzyme, a distinction is made between two types of immunological determination. When the labelled molecule is the antigen or hapten, reference is made to "determination by competition" or "determination in a limited reagent quantity". In this case, the principle of the determination is based on the competition between the labelled hapten or antigen on the one hand and the antigen or hapten on the other with respect to a limited number of antibody sites. Thus, for immunological determination, it is necessary to establish a calibration curve by reacting constant quantities of antibodies and labelled molecules of antigen or hapten in limiting quantities with variable hapten or antigen quantities. After establishing the antigen - antibody equilibrium, a fractionation method is used on the mixture, which makes it possible to separate the labelled complex from the antigen or hapten remaining free in solution and by measuring the enzymatic activity of the bonded fraction (complex) or the free fraction (labelled hapten or antigen), it is possible to determine the activity corresponding to the different antigen or hapten concentrations and plot the calibration curve. Once this curve has been plotted, it is possible to determine the antigen or hapten concentration of a sample by applying the same procedure and by referring to the calibration curve to obtain the hapten or antigen concentration corresponding to the enzymatic activity found during the determination.
When the labelled molecule is the antibody, reference is made to "immunometric" methods or to determination "with reagent excess". There are several variants to these methods and the three most important of these are described below.